We propose to study how biological information, in the form of specific nucleobases, is recognized and transferred accurately in cellular pathways. Errors in this information transfer lead to carcinogenesis and to drug resistance, and so understanding how they arise, and how to modulate them, can have direct implications in medical treatment of cancer and infectious disease. Our approach to studying these biological processes is to develop and study new DNA and RNA base analogs that modulate electrostatics and shape in a systematic way. We will use sets of these molecules to probe steric and electronic effects in four nucleobase-dependent processes, including the uptake and metabolism of specific nucleosides into cells; the polymerase-dependent copying of DNA and RNA sequences; protein-mediated RNA-RNA recognition in RNA interference; and specific recognition of damaged nucleobases. All of these require that the biological systems can accurately discriminate one nucleobase from others; we aim to study how this occurs. During the recent funding period we have made strong progress in understanding how varied classes of DNA polymerases identify correct versus incorrect nucleotides. This has led to new hypotheses concerning active site rigidity and flexibility, and how repair enzymes differ from replicative ones. Moreover, using new RNA base analogs we have generated exciting preliminary data on how siRNAs recognize and downregulate specific mRNAs. Finally, we have developed new analogs of damaged DNA bases, and have begun to use them to study mutagenesis and repair. In the near term covered by this proposal, we plan to test several new hypotheses for nucleotide recognition. Our specific aims include (1) study of the origins of sequence recognition in RNA interference; (2) researching the mechanisms of kinase-mediated nucleotide uptake and metabolism; (3) investigation of mismatch and damage recognition by new classes of polymerases. We expect that this work will lead to important basic insights into the chemistry and biology of nucleobases, and possibly to new diagnostic and therapeutic strategies involving nucleosides and nucleic acids. PUBLIC HEALTH RELEVANCE: We propose to study how the four nucleic acid bases of DNA and RNA are discriminated from one another in the biological pathways that involve this genetic information. This will be done by design and study of modified versions of these bases, and their function in these pathways. The work is important because it provides basic information about how mutations arise in DNA, and suggests new strategies for increasing activity and lowering toxicity of potential nucleotide- based drugs.